Team:PennState/diauxie/Strategies

We explored two options to make a glucose-independent, xylose-inducible system: 1) via the protein CRP*, and 2) by selectively engineering the promoter region sequence.

The first strategy was the main focus of the 2007 Penn State iGEM team. This approach used a mutated version of the protein CRP called CRP*, which acts as CRP bound to cAMP. This means that our system would always be “turned on” when xylose is present, even if glucose is also present. The problem with this approach is that CRP* is not specific to the xyl operon; it also regulates transcription in many other locations. Expressing CRP* in the cell can lead to having other systems turned on or off irregularly, possibly having toxic consequences.

This year we focused on constructing and characterizing engineered alterations of the xyl promoter region. The operator controlling genes for xylose metabolism are dependent on binding both the CRP-cAMP and XylR-xylose activated complexes. We attempted to engineer the promoter region so only XylR transcriptional control remained. To accomplish this, we first scrambled the CRP binding site DNA by random base changes. With this sequence deactivated, the possibility of CRP binding was eliminated; this is also significant because CRP may repress transcription when bound in the operator region without cAMP. The second alteration was to strengthen the RNA polymerase binding sites to compensate for the loss of CRP promotion. The binding affinity was strengthened by changing a few bases in the polymerase binding region. This was done in steps so that the regions began to resemble a consensus sequence for E. coli RNA polymerase binding. Depending on the mechanism(s) of interaction between XylR and RNA polymerase components, too strong of a promoter may result in high levels of constitutive expression (in the absence of xylose), which is undesirable.

Xyl Operon

Four different altered promoter regions were ordered from GeneArt. Promoter “PN” was synthesized as the wild-type promoter sequence with biobrick ends. This promoter is necessary for testing comparisons with altered region promoters. Promoter P1 was ordered as the natural promoter region with a scrambled CRP binding site. The P1 promoter should show weak RNA polymerase binding and transcription and will help to further elucidate the roles of CRP and XylR in xyl promoter expression. The “medium strength” promoter P3 contained a scrambled CRP binding site along with an altered RNA polymerase binding site, the latter changed to be a combination of the wild-type and consensus binding sequences. Finally promoter P5 contains a scrambled CRP binding site and an RNA polymerase binding site matching the consensus sequence.

To test the right-facing promoters we cloned RBS/GFP (E0240) downstream of each sequence. Fluorescence readings were used to measure the levels of induction.

@@ Line 176: / Line 176: @@
 <p>The first strategy was the main focus of the 2007 Penn State iGEM team.  This approach used a mutated version of the protein CRP called CRP*, which acts as CRP bound to cAMP.  This means that our system would always be “turned on” when xylose is present, even if glucose is also present. The problem with this approach is that CRP* is not specific to the xyl operon; it also regulates transcription in many other locations. Expressing CRP* in the cell can lead to having other systems turned on or off irregularly, possibly having toxic consequences.</p>
-<p>This year we focused on constructing and characterizing engineered alterations of the xyl promoter region.  The operator controlling genes for xylose metabolism are dependent on binding both the CRP-cAMP and XylR-xylose activated complexes.  We attempted to engineer the promoter region so only XylR transcriptional control remained.  To accomplish this, we first scrambled the CRP binding site DNA by random base changes.  With this sequence deactivated, the possibility of CRP binding was eliminated; this is also significant because CRP may repress transcription when bound in the operator region without cAMP.  The second alteration was to strengthen the RNA polymerase binding sites to compensate for the loss of CRP promotion.  The binding affinity was strengthened by changing a few bases in the polymerase binding region.  This was done in steps so that the regions began to resemble a consensus sequence for E. coli RNA polymerase binding. Depending on the mechanism(s) of interaction between XylR and RNA polymerase components, too strong of a promoter may result in high levels of constitutive expression (in the absence of xylose), which is undesirable.</p>
+<p>This year we focused on constructing and characterizing engineered alterations of the xyl promoter region.  The operator controlling genes for xylose metabolism are dependent on binding both the CRP-cAMP and XylR-xylose activated complexes.  We attempted to engineer the promoter region so only XylR transcriptional control remained.  To accomplish this, we first scrambled the CRP binding site DNA by random base changes.  With this sequence deactivated, the possibility of CRP binding was eliminated; this is also significant because CRP may repress transcription when bound in the operator region without cAMP.  The second alteration was to strengthen the RNA polymerase binding sites to compensate for the loss of CRP promotion.  The binding affinity was strengthened by changing a few bases in the polymerase binding region.  This was done in steps so that the regions began to resemble a consensus sequence for <em>E. coli</em> RNA polymerase binding. Depending on the mechanism(s) of interaction between XylR and RNA polymerase components, too strong of a promoter may result in high levels of constitutive expression (in the absence of xylose), which is undesirable.</p>
 <h4>Xyl Operon</h4>